Isoelectric focusing is the recommended method and the one used at Mayo Clinic, Dr. Willrich said. It requires running the samples in pairs with the CSF and serum next to each other because some bands are pres-ent in both CSF and serum and would not be considered unique bands in CSF.
In Fig. 2 is an example of a positive case that has at least 10 unique bands in CSF that are not present in serum. “This [isoelectric focusing] is the gold standard method for oligoclonal banding detection,” she said.
Mayo Clinic’s laboratory receives multiple samples daily that require oligoclonal banding testing. The serum and CSF samples are paired manually. Samples are built in the laboratory information system with four patients per gel, along with a negative and positive control.
“First we dilute the specimens by pipetting them onto gels,” Dr. Willrich said. “Then they are electrophoresed for 45 minutes.” After that initial electrophoresis, specimens are transferred with simple pressure to a membrane (the “blotting” stage), where they remain for 30 minutes. Next is the staining process, “where you use blocking solution for about an hour to prevent nonspecific binding of proteins,” and then stain. Three medical laboratory scientists read the gels and the number of bands.
The full isoelectric focusing process takes about four hours. “It’s entirely manual, and it’s hard to automate a process like this in the lab,” she said. The medical laboratory scientists stand and move from one area to another all day, “so they consider it a very demanding bench.” The monthly test volume as of January 2026 exceeded 1,700 cases.
Bands are counted in the CSF samples, and any bands found in the serum sample that line up with bands found in the CSF sample are then subtracted. A negative and positive control must be present for the gel to be acceptable. “We see a lot of variations,” Dr. Willrich said. “Someone can read 11 bands, 12 bands, 13 bands, and then we go with an average of the number of bands that were counted.”
The problems include overstaining, understaining, poor application, uneven electrophoresis, inadequate pressing, and bubbles (Fig. 3). “They prevent us from reading that accurately, and those gels are repeated,” she said, adding to the cost and turnaround time.
Mayo Clinic’s oligoclonal banding test report would include, as an example, the following: CSF bands, 11; serum bands, 3; CSF unique bands, 8; reference interval, ≥ 2; interpretation, positive/abnormal; and additional interpretive comments.
“Some people do not report the number of bands they see; they only report the delta or they only call it positive,” Dr. Willrich said, noting that reporting is not standardized. One study of clinical chemists in all 13 Canadian clinical laboratories performing CSF OCB analysis revealed that “Profound variation exists in processes, reporting, and interpretation of CSF OCB and associated tests and indices across Canadian clinical laboratories” (Higgins V, et al. Clin Biochem. 2023;116:105–112).